Morphometric and statistical analyses describing the In utero growth of human epidermis.

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THE ANATOMICAL RECORD 222201-206 (1988)
Morphometric and Statistical Analyses Describing
the In Utero Growth of Human Epidermis
CAROLYN A. FOSTER, JOHN F. BERTRAM, AND KAREN A. HOLBROOK
Department of Dermatology 1, Division of Immunobiology, University of Vienna Medical
School, Vienna A-1090 Austria (C.A.F.);Department of Anatomy, University of Melbourne,
Victoria 3052, Australia (J.F.B.)Departments of Biological Structure and Medicine
(Dermatology); University of Washington, School of Medicine, Seattle, WA 98195 (K.A.H.)
ABSTRACT
Epidermal development of human embryonic and fetal skin from the
lower limb was studied using morphometric and statistical methods. Epidermal growth,
as defined by an increase in epidermal thickness and the number of cell layers,
occurred in three distinct stages during the first and second trimesters. The first
growth spurt occurred between 5 and 13 weeks estimated gestational age (EGA)and
was followed by a plateau phase with little change in epidermal thickness from 14
to 21 weeks, after which the epidermis began to increase in height again. The periderm
reached its maximal height by approximately 13 weeks EGA, and by 25 weeks was
shed into the amniotic fluid. Thus, within a five-month period (5 to 25 weeks EGA)
the epidermis changed from a single cell layer < 10 pm thick to a 10 to 12-celllayer,
keratinized epithelium > 60 pm thick. In contrast, epidermis from adult lower limb
consisted of about 25 cell layers and was almost 75 pm in thickness. The age-related
differences in epidermal thickness probably reflect changes in cell size and shape
more than changes in the directional movement (apically vs. laterally) of proliferating
keratinocytes, because the addition of cell layers throughout development was relatively constant. During the plateau phase, when there is a rapid increase in fetal
growth rate, the suprabasal keratinocytes become more flattened, thereby allowing
for the addition of new cell layers while maintaining a relatively constant epidermal
thickness. The loss of glycogen reserves, along with other intrinsic cellular changes,
probably contributes to the flattening effect. However, the internal expansion of growing tissues also may exert a mechanical pressure that could stretch the skin passively
and influence epidermal structure.
The skin is not only the largest organ of the body but
is also one of the first to form during development.
Within 1 month after zygote formation the human embryo is covered by a bilayered anlage of skin consisting
of a simple epithelium (epidermis) overlying a thin, hydrated mesenchyme (dermis). These ectodermal and
mesodermal derivatives differentiate into a miniaturized replica of adult skin by 6 months gestation. Numerous investigators have documented the ontogeny of
human skin by morphological and biochemical criteria
(Moll et al., 1983; Dale et al., 1985; Smith et al., 1986;
reviews by Breathnach, 1971; Holbrook, 1979; Holbrook
and Hoff, 1984). However, there appears to be no quantitative information on dimension within the skin compartment during development.
The density of certain epidermal cells (e.g., Langerhans cells) is usually referenced to a unit area of skin.
Except for certain diseases affecting skin thickness
(Haftek et al., 19831, this is considered to be an acceptable method of comparing cell numbers in adult
skin. Epidermal cell numbers based on surface area can
be similarly determined in embryonic and fetal skin,
but without three-dimensional measurements these data
could be misleading because the cutaneous compartment is continuously expanding and differentiating
throughout gestation.
0 1988 ALAN R. LISS, INC.
To better understand the Langerhans cell population
in developing skin from a two- and three-dimensional
perspective (Foster et al., 1986; Foster and Holbrook,
in preparation), the thickness of human eipdermis was
evaluated a t progressive stages of gestation. Because
the surface contours of developing epidermis are highly
variable (Holbrook and Odland, 1975), a morphometric
(digitizing) technique was chosen to determine epidermal height, and the data were statistically analyzed.
This study provides the first mathematical description,
in the form of polynomial equations, of human epidermal growth in utero and suggests a pattern of coordinated events that culminate in the formation of a
stratified, keratinized epithelium.
MATERIALS AND METHODS
Specimen Collection
Human embryonic and fetal tissue was provided
through the courtesy of Dr. Thomas Shepard, Director
Received January 12, 1988; accepted March 7, 1988.
Address reprint requests to Dr. Karen A. Holbrook, Department of
Biological Structure SM-20, University of Washington, School of Medicine, Seattle, WA 98195.
202
C.A. FOSTER, J.F. BERTRAM, AND K.A. HOLBROOK
of the Central Laboratory for Human Embryology at
the University of Washington. Consent forms and collecting practices were approved by the University of
Washington Human Subjects Review Committee. Tissue was obtained from the lower limbs of 38 human
embryos and fetuses, ranging in age from 5 to 25 weeks.
Estimated gestational age (EGA = fertilization age)
was determined from patient histories and fetal measurements (crown-rumpand foot length). Specimens that
were 10 weeks EGA or younger were designated as
embryonic, while those older than 10 weeks were termed
fetal. Skin from three healthy adult volunteers (30-35
yr) was obtained from the ventral thigh by punch biopsy.
Tissue Processing
Pieces of skin were immersed in half-strength Karnovsky’s fixative (Karnovsky, 1965) overnight at 4”C,
rinsed in 0.1 M cacodylate buffer, then post-fixed for
an additional hour in 1%OsO, in distilled water at room
temperature. After a brief wash in water, the tissue
was stained en bloc with 1%aqueous uranyl acetate for
1 hr, dehydrated in a graded series of ethanols into
propylene oxide, and embedded in Epon 812 (Luft, 1961).
Semi-thin (1 p m ) sections were stained with toluidine
blue (Richardson et al., 1960). Ultrathin sections (800
A) were stained with aqueous uranyl acetate and lead
citrate (Reynolds, 1963) and examined with a Philips
201 transmission electron microscope.
area of the first sections cut from the block face. Linear
compression was calculated as the ratio of these two
areas. Two blocks analyzed from each specimen at each
of three stages (6,10,20 weeks EGA) showed that there
was no measurable compression of the sections.
Enumeration of Epidermal Cell Layers
The mean number of epidermal cell layers was counted
in toluidine blue-stained, 1 pm-thick sections of Eponembedded skin from three lower limbs of 34 individuals
(5 to 25 weeks EGA and three adults). Ultrathin sections of keratinized epidermis were examined in the
transmission electron microscope to delineate the number of layers in the stratum corneum.
Calculation of Epidermal Height
To estimate epidermal thickness, approximately eight
sections of skin per specimen, usually representing two
blocks of tissue, were photographed under oil immersion at x 40 on 35 mm film (Panatomic-X) with a Zeiss
photomicroscope. Successive frames of film were projected onto the digitizer tablet of a microcomputer-based
morphometric facility (Sundsten and Prothero, 1983),
and the contours of a constant length of interfollicular
epidermis, with and without the peridermal layer, were
traced manually with a “mouse.” The coordinates of
each contour were stored on floppy disks in a North
Star Horizon microcomputer and cross-sectional areas
were computed using a software package (MORPHO)
Assays for Shrinkage and Compression
for morphometric data (Prothero and Prothero, 1982).
Epidermal height was determined by dividing the
Experiments were conducted to determine if tissue
processing and sectioning might affect the epidermal area of each contour by 0.24 mm (the constant length
dimensions being measured. As described elsewhere of epidermis measured in each frame of 35 mm film)
standard deviation
(Foster, 1987), embryonic and fetal skin was removed and calculating the mean value
from the lower limb and divided into two groups: 1) for each specimen.
whole skin with subcutaneous tissue removed, and 2)
Applied Regression Analysis
skin with most of the dermis peeled away. Eight pieces
The
relationship
between age and epidermal dimenof skin per group were cut into square or rectangular
pieces (3-4 mm2) and measured en face, both before sions was determined using applied regression analysis
and after fixation in Karnovsky’s fixative, then again (Kleinbaum and Kupper, 1978). Least-squares analysis
after polymerization in Epon. Changes in linear di- was used to minimize the deviation between the obmensions due to tissue processing were calculated from served and fitted points and, thus, to describe the bestthe following equation: postembedment diameterlpre- fitting curve mathematically. The appropriate order
fixation diameter x 100 = % of original linear dimen- (first, second, or third) for the polynomial model was
determined by calculating the test statistic (F = MS
sion.
Although it is known that tissue processing through regressiodMS residual) and testing the null hypothesis
primary fixation and Epon embedment causes differ- H, = no significant lack of fit of the assumed model.
ential, age-dependent shrinkage of skin during ontogRESULTS
eny (Foster, 1987), it was difficult to correct for these
Morphometric and Statistical Measurements of Epidermal
effects on measurements of epidermal height without
Thickness
the risk of grossly distorting the data. For instance, the
Epidermal growth in developing human skin, as dedermis had a profound influence on differential shrinkage in Karnovsky-fixed skin, as determined by changes fined by the rate of increase (i.e., slope) in epidermal
in the linear dimensions of skin pieces that were meas- height, was measured by two approaches: calculating
ured en face; however, it was technically impractical to epidermal thickness utilizing a morphometric, digitizmeasure the depth of skin in these assays. In contrast, ing technique and counting the number of cell layers.
postfxation processing through Epon accounted for about Within a 5-month period (5 to 25 weeks EGA) the ep4% of the total net shrinkage (Foster, unpublished ob- idermis changed from a single cell layer < 10 pm-thick
servations), and these changes were not age-dependent. to a 10 to 12-cell layered, keratinized epithelium > 60Therefore, measurements of epidermal height were ad- pm thick (Figs. 1-3). In contrast, epidermis from adult
justed for 4% linear shrinkage because it was reason- lower limb had about 25 cell layers and was almost 75
able to assume that the epidermal compartment might ym in thickness (Figs. lg, 2).
Epidermal growth of the lower limb occurred in three
also change by this amount.
Tissue compression due to sectioning was assayed for different stages during embryonic and fetal developby comparing the area of the Epon block face to the ment. The first growth spurt occurred between 5 and
*
203
IN U T E R O GROWTH OF HUMAN EPIDERMIS
14 wks
1
17 wks
rc
Adult
Fig. 1. Photomicrographs (left panel) of toluidine blue-stained sections
(1 km-thick) of Epon-embedded embryonic fetal and adult skin, with
corresponding profiles of digitized epidermis (right panel.) P, periderm
a-g X320.
204
C.A. FOSTER, J.F. BERTRAM, AND K.A. HOLBROOK
u
-
Epidermis w i t h periderrn
t Epidermis w i t h o u t periderrn
70
Iy
=
-137.67
+
x
E
5 2 . 8 2 6 ~- 5 . 9 5 9 ~+ ~0 . 2 4 3 ~ ~
( R = 0.991)
60-
I-
I
W
0
5040-
-I
2
30-
Ly
I
g
I
r/u
-L
w
-
*-A&'
8
12
10
14
18
16
20
-+IAd"lt
2
4
22
Y
a
5
W
E
k
7
111
y
= -48.832
53
W
6-I
'
I
6
*
I
I
'
9
8
I
.
'
I
10
11
I
'
12
'
I
13
Fig. 4. Least-squares plot and equation describing the relationship
between epidermal height (pm) and EGA in weeks (6 to 13).
Fig. 2. Plot of epidermal height (pm) 2 standard deviation showing
measurements with the periderm layer, without periderm, and periderm
only (see Table 1) against EGA in weeks (6 to 23). Adult values (with
the stratum corneum) are included.
I
.
r
'
ESTlPlATED G E S T A T I O N A L AGE ( w k s )
ESTIMATED GESTATIONAL AGE (Wks)
W
I /
10,
5
---&-
0
6
20-
501
-
I
14
.
I
16
+
*
.
1 9 . 0 3 6 ~- 1 , 184x2
( R = 0.931)
I
18
.
+
0.024~
0
I
20
8
22
1
7
24
-
26
ESTIMATED GESTATIONAL AGE (wks)
4-
-
Fig. 5. Least-squares plot and equation describing the relationship
between epidermal height (pm) and EGA in weeks (14 to 25).
2-
rt
0 7 '
4
6
1
'
I
8
.
I
10
'
.
12
I
14
'
9
16
.
I
'
8
18 20
1
,
'
8
'
'
22 2 4 26
E S T I H A T E D G E S T A T I O N A L AGE (wks)
Fig. 3. Least-squares plot and equation describing the relationship
between number of epidermal cell layers and EGA in weeks (5 to 25).
13 weeks EGA when the epidermis increased in height
from < 10 pm to about 66 pm, with a slope of 7.5 (Fig.
2); the number of cell layers increased from one to four
(Fig. 3). The periderm (the outermost epidermal layer)
reached its maximal height by 12.5 to 13.5 weeks (Figs.
lc, 2, Table l),then began to decline as the cells entered
the complex bleb stage described by Holbrook and Odland (1975)and expanded laterally. Periderm regression,
marked by a decrease in periderm thickness, loss of
glygogen, and formation of a cornified cell envelope, was
most dramatic between 13 and 14 weeks (Fig. lc,
d), as reflected by the abrupt decrease in epidermal
thickness from about 66 pm to 52 pm (Fig. 2, Table 1).
Between 14 and 21 weeks, designated the "plateau
phase," the epidermis increased from four to six layers
(Fig. 3) but essentially remained the same thickness
(about 50 pm). The second growth spurt occurred after
21 weeks when the epidermis began to increase in height
(Fig. 2), undergo interfollicular keratinization (Fig. 10,
and accumulate more cell layers in the stratum intermediudspinosum and stratum corneum. As indicated
by a slope of 3.5, the increase in epidermal thickness
between 21 and 25 weeks was more gradual than during
the first growth phase before 13 weeks (Fig. 2).
These data were tested by applied regression analysis
and plotted as a function of age. The cubic equation
y = -137.67 + 52.826~- 5.959~' ,0.243x3(R =
0.991), where y = epidermal height in micrometers and
x = EGA in weeks (Fig. 4), best described the relationship between age and epidermal thickness during the
first trimester (6 to 13 weeks). Epidermal growth during the second trimester, from 14 to 25 weeks (Fig. 5),
was defined by the equation y = -48.832 + 19.036~1.184~2+ 0.024~3(R = 0.931). The relationship between EGA and number of cell layers also was curvilinear. The equations y = 3.81 - 0 . 5 4 6 ~+ 0.047~' 0 . 0 0 1 (R
~ ~= 0.989) and y = - 3.944 + 1 . 4 0 5 ~
- 0.094~~
0.002~
(R~ = 0.988) where y = number of cell layers
and x = EGA in weeks, best described the relationship
between 5 and 25 weeks EGA, with (plot not shown)
+
+
205
IN UTERO GROWTH OF HUMAN EPIDERMIS
TABLE 1. Epidermal height measurementsat progressive stages of human
gestation
Estimates
(wk)
Gestational age
no. of specimens
6
7
8
9
10
11
13
14
16
17
21
23
Adult
4
2
3
3
3
2
3
4
Epidermal thickness + S.D. (pm)
With periderm
Without periderm
Periderm only
18.14 f 0.67
20.88 2 3.56
28.65 f 4.68
33.64 2 1.87
36.89 f 1.88
44.32 f 4.47
65.83 f 8.63
51.68 i 4.16
52.83 f 4.88
51.79 f 2.80
50.96 -t 3.12
60.11 f 6.06
74.46 f 8.94l
12.85 f 1.04
16.03 f 3.29
21.34 f 4.71
25.15 f 4.47
27.19 ? 2.70
31.36 -t 5.22
48.20 f 4.75
42.32 f 5.09
45.76 -t 2.34
47.32 _t 2.08
48.10 ? 4.16
56.13 ? 6.86
NA
5.28 2 0.36
4.88 c_ 1.41
7.28 f 1.56
8.52 f 2.91
9.77 f 2.39
13.72 f 0.83
16.69 f 3.64
7.86 f 2.12
7.12 f 2.60
4.47 -t 2.91
2.86 -t 1.04
1.72 ? 0.93
NA
'Including a stratum corneum.
and without adult values, respectively (Fig. 3). It should
be emphasized that the above equations do not necessarily predict outside of the designated age range
for a given regression plot, e.g., 6 to 13 weeks EGA in
Figure 4.
Epidermal measurements excluding the periderm also
were calculated from digitized contours (Fig. 2). The
same basic pattern of increasing epidermal thickness
was apparent. However, unlike the trend including periderm measurements, there was no statistically signficant decrease in epidermal thickness after 12 to 13
weeks. The periderm steadily increased in thickness,
from 5 pm at 6 weeks to almost 17 pm by 12.5 to 13.5
weeks, then gradually decreased in height (Fig. 3, Table
1). By 23 weeks the peridermal layer was less than 2
pm in thickness, and by 25 weeks the periderm cells
were shed into the amniotic fluid.
DISCUSSION
Quantitative, morphological techniques were used to
analyze epidermal development of the lower limb. Development was defined as an increase in epidermal
thickness and accumulation of cell layers. Three distinct growth stages between 5 and 25 weeks EGA were
identified. Although previous investigators have described human embryonic and fetal development both
morphologically and biochemically (Moll et al., 1983;
Dale et al., 1985; Smith et al., 1986; reviews by Breathnach, 1971; Holbrook, 1979; Holbrook and Hoff, 1984),
this is the first quantitative study in humans of epidermal dimensions at progressive stages during the first
two trimesters of embryonic and fetal development.
The observed trends in epidermal growth seem to
correlate with the two main phases of gestational maturation (Moore, 1982), i.e., organogenesis (embryonic
period) and rapid body growth along with differentiation of established organs (fetal period). Most of the first
growth spurt (5 to 13 weeks), when the epidermis undergoes its greatest increase in thickness, coincides with
the initial development of the main organ systems (organogenesis) with little body growth. Conversely, the
epidermal height remains essentially unchanged for almost 2 months (14 to 21 weeks) during the plateau
phase when the rate of body growth is very rapid (Moore,
1982), including 5-fold increase in surface area of the
lower limb (calculated from Klein and Scammon, 1930).
During this latter stage, some of the keratinocytes may
move laterally to accomodate the increased surface area,
but many are also growing in a downward direction as
they give rise to the epidermal appendages (review by
Holbrook, 1979). Changes in body surface area, cell
structure, and mitotic activity are among the factors
that could influence the pattern of epidermal growth
during embryonic and fetal development.
The differences in epidermal thickness would appear
to reflect changes in cell size and shape more than
changes in the directional movement (apically versus
laterally) of proliferating keratinocytes because the addition of cell layers is relatively constant between 5 and
21 weeks. In general, embryonic and early fetal keratinocytes are plump and balloon-shaped with large
amounts of glycogen (Holbrook and Odland, 1975). By
the second trimester, suprabasal keratinocytes have expanded and flattened in parallel with cells of the periderm, while basal cells have become less cuboidal, more
columnar, and largely depleted of their glycogen content. Lateral expansion and regression of periderm cells
after about 13 weeks accounts for a considerable decrease in epidermal height; however, the basic pattern
of increasing epidermal thickness is still reflected in
measurements excluding the peridermal layer. The
flattening of epidermal cells during the plateau phase
may be a mechanical response, in part, to the rapid
internal expansion of growing tissues, thereby allowing
for the addition of new cell layers while maintaining a
relatively constant epidermal thickness. However, intrinsic cellular changes, particularly the loss of glycogen reserves from the suprabasal cells, would probably
contribute more to the flattening of keratinocytes.
Although mitotic activity must play an important role
in determining the pattern of epidermal development,
little is known about the dynamics of keratinocyte proliferation during ontogeny. A few investigators have
observed a steady decrease in the labeling index of human embryonic and fetal epidermal cells from 26% at
8 weeks EGA to 4% by 16 to 19 weeks (calculated from
Gerstein, 1971; Stern, 1974; Bickenbach and Holbrook,
1987). The high labeling index in humans between 8
and 12 weeks EGA is likely due to thymidine incorporation by cells in basal, intermediate, and periderm
206
C.A. FOSTER, J.F. BERTRAM, AND K.A. HOLBROOK
layers (Stern 1974; Bickenbach and Holbrook, 1987).
By 4 to 5 months labeling of fetal keratinocytes is restricted to the basal layers, and the labeling index appears to have stabilized to a value similar to that of the
adult (5.5%)(calculatedfrom Epstein and Maibach, 1965;
Lachapelle and Gillman, 1969; Weinstein and Frost,
1969; Heenan and Galand, 1971; Flaxman and Chopra,
1972; Allegra and Panfilis, 1974; Gelfant, 1982; Stern,
1974). Thus, based on published data and the present
observations, we conclude that an inverse relationship
exists between the mitotic index and the increase in
both epidermal thickness and number of cell layers.
The trends that have emerged from this study of epidermal development may help to provide a better understanding of the complex, interrelated events that
culminate in the formation of a stratified and keratinized epithelium. Although the data reported herein are
limited to one region of the body, it has been demonstrated that the thickness of the epidermis is reasonably
consistent among most body regions at developmental
ages ranging from 45 days EGA to 22 weeks. The head,
palms, and soles, however, are exceptions showing accelerated differentiationcompared with the other regions,
particularly during the second trimester (Holbrook and
Odland, 1980).Thus the measurements for the thigh may
be considered representative for the body in general.
Flaxman, B.A., and D.P. Chopra 1972 Cell cycle of normal and psoriatic
epidermis in vitro. J . Invest. Dermatol., 59t102-105.
Foster, C.A. 1987 Differential effects of tissue processing on human
embryonic and fetal skin. Anat. Rec., 218;355-358.
Foster, C.A., RA. Holbrook, and A.G. Farr 1986 Ontogenyof Langerhans
cells in human embryonic and fetal skin: Expression of HLA-DR and
OKT-6 determinants. J. Invest. Dermatol., 86:240-243.
Gelfant, S. 1982 “Of mice and men”: The cell cycle in human epidermis
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Haftek, M., M. Faure, D. Schmitt, and J. Thivolet 1983 Langerhans cells
in skin from patients with psoriasis: Quantitative and qualitative
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with aromatic retinoid adminstration. J. Invest. Dermatol., 81:lO14.
Heenan, M.A.H., and P. Galand 1971 Cell population kinetics in human
epidermis: In vitro autoradiographic study by double l a b e h g method.
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Holbrook,K.A. 1979 Human epidermal embryogenesis. Int. J. Dermatol.,
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Holbrook, KA., and M. Hoff 1984 Structure of the developing human
embryonic and fetal skin. Semin. Dermatol., 3t185-202.
Holbrook, K.A., and G.F. Odland 1975 The fme structure of developing
human epidermis: light, scanning, and transmission electron microscopy of the periderm. J . Invest. Dermatol., 65t16-38.
Holbrook, KA., and G.F. Odland 1980 Regional development of the human epidermis in the first trimester embryo and the second trimester
fetus (ages related to the timing of amniocentesis and fetal skin
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Karnovsky, M.J. 1965 A formaldehyde-glutaraldehyde furative of high
osmolarity for use in electron microscopy. J. Cell Biol., 27t137A.
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ACKNOWLEDGMENTS
other multivariable methods. Duxbury Press, North Scituate, Mass.
pp. 113-130.
We are grateful to Drs. John Sundsten, Paul Samp- Lachapelle,
J.M., and T. Gillman 1969 Tritiated thymidine labelling of
son (Department of Statistics), Mr. James Holbrook
normal human epidermal cell nuclei: A comparison, in the same
subjects, of in vivo and in vitro techniques. Br. J . Dermatol.,
(NOAA),and Ms. Mary Pat Larson for generously shar81t603-616.
ing their expertise in computers and statistical analyJ.H. 1961 Improvements in epoxy resin embedding methods. J.
sis. We wish to thank Mr. Robert Underwood and Mrs. LUR,Biophys.
Biochem. Cytol., 9:409-414.
Mary Hoff for their invaluable technical assistance, and Moll, R., I. Moll, and W. Wiest 1983 Changes in the pattern ofcytokeratin
polypeptides in epidermis and hair follicles during skin development
Drs. John Prothero and Pritinder Kaur (Fred Hutchin human fetuses. Differentiation, 23:170-178.
inson Cancer Research Center) for helpful suggestions.
K.L. 1982 The Developing Human. W.B. Saunders Co., PhilaThis study was suported by grants HD 17664 (K.A.H.), Moore,
delphia, pp. 366-374.
AR 21557 (K.A.H.)from the National Institutes of Health Prothero, J., and J . Prothero 1982 Three-dimensional reconstruction
from serial sections. I. A portable microcomputer-based soRware
and Public Health Service National Research Service
package in Fortran. Comput. Biomed. Res., 15:598-604.
Award 2 T32 GMO7270-09 (C.A.F.)
Reynolds, E.S. 1963 The use of lead citrate at high pH as an electron
opaque stain in electron microscopy. J. Cell. Biol., 17:208-213.
Richardson, K.C., L. Jarret, and E.H. Finke 1960 Embedding in epoxy
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